Toxicants are a common source of stress to aquatic ecosystems, often acting in combination with other pressures such as river channel alterations, eutrophication, changes to water temperature and pH and invasive species.

Unravelling the combinations of different chemicals able to cause toxic stress to freshwaters is a really complex process, since such ‘cocktails’ can contain mixtures of substances drawn from 10⁶⁰ possible chemicals.

Ever tried to visualise such a huge number as ten to the power of sixty? SciencePie suggest a thought experiment that might help. Take 10⁶⁰ grains of sand and pile them up over the entire area of the United States of America. This would lead to a pile of sand with a height of around 15,000,000 times the diameter of the observable universe.

Do we have to analyse such a myriad of chemicals in order to understand toxic stress? Of course not. However, we need efficient and accurate tools to identify the chemicals that cause major stresses on aquatic organisms in a water body or river basin. If water quality monitoring and environmental management is only based on a pre-selected range of chemicals (such as the Water Framework Directive priority pollutants) we risk ignoring those chemicals that actually cause ecosystem effects. In turn, this approach risks focusing our monitoring and management resources on compounds of minor relevance.

What is Effect-Directed Analysis?

Effect-Directed Analysis (EDA) is one of the major approaches used to support the identification of toxic compounds at a specific site by combining chemical and biological analytical tools. It uses fractionation, a laboratory process which separates chemical mixtures allowing the determination of its different components. Although EDA has been used by environmental laboratories for over twenty years, a comprehensive compilation of EDA tools and recommendations for their efficient use was – until recently – missing.

Now, a group of 26 leading experts from Europe, U.S.A. and Canada have joined efforts to publish an in-depth overview on EDA over 46 pages of the journal Science of the Total Environment. This review provides a conceptual framework for the integration of EDA into water quality monitoring, and considers toxicant identification in diverse matrices (water, sediments and biota). Their work has been supported by the projects SOLUTIONS, EDA-EMERGE and the NORMAN Network on emerging pollutants.

Bioassays: unravelling chemical cocktails using biological indicators

As an effect-based approach, EDA is driven by the measurable effects of chemical mixtures extracted from these matrices using bioassays. Bioassays are biological analytical tools that detect effects of individual compounds and environmental mixtures on organisms and cellular systems, so called in vivo (on living organisms) and in vitro (on microorganisms and cells isolated from their normal environment) studies. In vivo bioassays typically use representatives of major organism groups also addressed as Biological Quality Elements in the Water Framework Directive including algae, invertebrates (e.g. Daphnia) and fish (e.g. zebrafish embryos).

Daphnia as test organisms. Images: Andre Künzelmann.

Zebrafish as test organisms. Images: Andre Künzelmann.

In vitro bioassays often directly address specific modes of action including the metabolism of chemicals, hormonal disruption, inhibition of specific enzymes, DNA damage or adaptive stress responses. All these assays represent links in adverse outcome pathways following the concept that key events at the cellular level (e.g. the reaction of a chemical with DNA) may be mitigated by adaptive stress responses or, if repair capacity is exceeded, may lead to cascading effects on organisms, populations and even ecosystems.

Getting meaningful results from small samples

An EDA study typically involves the analysis of many samples and fractions which are only available in small amounts. Therefore, in addition to the biological response requirements of a sample, it also ideally needs to be processed at a fast rate (known as high throughput) and at low volumes. The new review lists the frequently used high throughput bioassays for EDA, along with their respective advantages and disadvantages, confounding factors, volumes and dosing formats.

Prioritisation, or listing in order of (in this case) toxic priority, of compounds contributing most to observed effects is the major task of effect-directed analysis. This may succeed only if the relative composition of a mixture of chemicals exposed to a test organism in the lab is similar to the mixture the organism would encounter in the field. The way in which extracts of sediments or water are dosed into bioassays very much determines the mixture taken up by the test organisms, and thus the priority setting.

In sediments, the mixture of chemicals to which a benthic organism is exposed is the result of partitioning (or otherwise stated: the ratio of concentrations) of all its components among sediment particles, water and the organism, while in the lab simple transfer by extraction and dissolution are predominant. In the overview paper, a thought experiment suggests that conventional dosing of sediment extracts may result in a strong bias in toxicant prioritisation, being recommended to load sediment extracts into silicone or other devices mimicking the natural conditions.

Different EDA tools: fractionation and mass spectrometry

In order to divide toxic chemicals and combinations from the bulk of inconspicuous ones, we need fractionation tools, typically chromatography, which reduce the chemical complexity of a sample in a smart way. That means we have to fractionate without losing our original toxicity; we need optimal selectivity for relevant chemical groups, and we should learn as much as possible about the properties of the chemicals ending up in the fractions. The overview paper brings together a great deal of the available research and experience to help design tailor-made fractionation procedures in the future.

When toxic effects (and the chemicals that cause them) are isolated in specific fractions, high-end analytical chemistry comes into play. High resolution mass spectrometry coupled to gas and liquid chromatography helps to identify the molecular formulas of the components in our toxic fractions. These formulas tell us the type and number of atoms involved in forming a molecule. However, they don’t tell us how these atoms are bonded to a chemical structure.

Many hundreds, even thousands of chemical structures with completely different properties and toxicities are possible, despite being described by the same molecular formula. The overview paper explains how it is possible to sequentially reduce the number of candidate chemical structures by comparing observations for the compounds in a sample such as chromatographic retention, ionization and fragmentation in mass spectrometry and toxic effects with predictions for candidate structures. This also involves a assemblage of computational prediction tools.

Clever SOLUTIONS for complex chemical problems

The marriage of advanced analytical and bio-analytical tools with rapidly developing computational prediction tools opens a very promising future for toxicant identification. Hopefully, this extensive overview paper can support this process and help to increase the applicability and success rate of effect-directed analysis in environmental monitoring.

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Features, interviews and analyses on freshwater conservation, science and policy.

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The blog was founded and edited between 2010-14 by the BioFresh project, an EU-funded project that built a global information platform for scientists and ecosystem managers with access to all available data describing the distribution, status and trends of global freshwater biodiversity.

Since 2014, the blog has been managed and edited by the MARS project – an EU-funded project which investigates how multiple stressors affect rivers, lakes and estuaries.

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